Melanosomes are tissue-specific intracellular organelles that function in the biosynthesis, storage, and transport of melanins in pigment cells of the eye and skin melanocytes. Melanosomes form within these cells through progressive developmental stages by the ordered delivery of specific protein contents from early endocytic organelles. Defects in melanosome formation cause oculocutaneous albinism (OCA), a condition associated with impaired vision and enhanced cancer susceptibility in the skin and eye. OCA is one of several systemic conditions associated with the Hermansky-Pudlak syndromes (HPS), genetic disorders in which melanosomes and other related tissue-specific organelles fail to form properly. Most forms of HPS and related disorders in mice result from mutations in genes that encode subunits of cytoplasmic multisubunit protein complexes involved in melanosome formation. Three of these complexes, AP-3, BLOC-1 and BLOC-2, function in trafficking distinct sets of transmembrane proteins between early endosomes and melanosomes. AP-3 is a coat protein that packages protein cargoes into endosome-derived vesicles, but the molecular functions of BLOC-1 and BLOC-2 are not known. We will exploit the genetic tools afforded by AP-3-, BLOC-1- and BLOC-2-deficient melanocytes to define endosome-to-melanosome anterograde and retrograde trafficking pathways in generalizable terms and define the molecular interactions that govern them. Preliminary evidence implicates VAMP7 and syntaxin 13, two SNARE family members that mediate membrane docking and fusion in the endosomal pathway, and the small regulatory GTPase Rab11 as functionally important in the BLOC-1 and/or AP-3 pathways. We hypothesize that BLOC-1, BLOC-2 and AP-3 function in concert with Rab11 to regulating recycling endosome membrane dynamics and SNARE-dependent membrane fusion with maturing melanosomes, and that syntaxin 13 and VAMP7 are components of the requisite SNARE complexes. We will test this general hypothesis in the following specific aims: 1. To test the hypothesis that the SNARE protein, syntaxin 13, is a component of the SNARE complex that delivers cargo to melanosomes by the BLOC-1-regulated pathway. 2. To test the hypothesis that the SNARE protein, VAMP7, is a component of the SNARE complex that links AP-3-dependent cargo delivery to melanosomes with BLOC-1-dependent cargo delivery. 3. To test the hypothesis that BLOC-2 functions in regulating SNARE-dependent fusion of early endosomal tubules with melanosomes. 4. To test the hypothesis that Rab11 effectors function as sensors of melanosome maturation by regulating the balance of anterograde and retrograde transport between endosomes and melanosomes. By defining these functions and pathways, we will further our understanding of the etiology of genetic disorders of organelle formation and provide insights into treatment regimens for HPS. PUBLIC HEALTH RELEVANCE: Hermansky-Pudlak syndrome is a group of genetic disorders that are common in Puerto Rico and are characterized by oculocutaneous albinism, excessive bleeding, and in many cases a fatal lung fibrosis and/or immune deficiency. The symptoms result from the failure to form certain specialized membrane structures, or organelles, within affected cell types. This proposal uses the melanosome, a target organelle within pigment cells, as a model system to understand how the products of the genes that are disrupted in Hermansky-Pudlak syndrome patients normally function to generate specialized organelles.